1. Field of the Invention
This invention generally relates to a method and apparatus for identifying or quantifying polypeptides. More specifically, this invention can be used to identify or quantify polypeptides that have been separated using a porous support, for example, using polyacrylamide gel electrophoresis (PAGE). The present invention can also be used to identify unseparated proteins from any other 1 or 2-dimensional array of proteins, for example, proteins absorbed onto a membrane, as in a tissue slice or organ blot, or from cells growing on a bacterial or tissue culture plate, or from a microtiter plate containing proteins from 1 or 2-dimensional gels or chromatography fractions. The technique involving separated polypeptides is especially useful in biochemical studies where the goal is to rapidly identify or quantify tens to hundreds of the most abundant proteins in a biological sample, while retaining some information about the starting molecular weight of the proteins that are identified. The technique that does not involve pre-separating polypeptides can be used to determine which proteins are the most abundant in different sections of any two-dimensional protein array. Relative quantification of separated proteins from one or more samples is accomplished by using stable isotope labeling of the proteins prior to separation, electroblotting, digestion and mass analysis.
2. Description of the Related Art
Biochemists often are interested in establishing the major protein constituents of biological samples, and commonly are interested in knowing which proteins have changed between an experimental sample and a control sample. In the last decade, gel electrophoresis followed by peptide mass fingerprinting (PMF) or tandem (MS-MS) mass spectrometry have become the methods of choice to perform these analyses. Typically, gel electrophoresis is performed to separate the proteins, and the whole gel is stained with Coomassie brilliant blue. After studying the gel pattern, the scientist determines which slices are to be analyzed. Manual or robotic methods are then used to excise the pieces of gel containing the protein(s) of interest. After several rounds of washing, the whole gel slice is separately digested with a protease, commonly trypsin, and the digestion products are extracted from each gel slice, concentrated, desalted, and submitted separately to mass spectrometric analysis. The mass spectra are often collected in a fully automated way, and special software is commercially available to make automatic protein identifications. The robots that perform the initial sample preparation steps are also commercially available, but are costly. In addition, the scientist must keep track of how each sample was related to the original gel during all of these steps, which introduces many opportunities for confusion. In many cases, the samples become accidentally contaminated with exogenous contaminants to differing degrees during these processes. These processes typically require several days to perform, including typically an overnight protease digestion step. Because these techniques are laborious and require a high degree of operator training, protein identifications of this kind are performed mainly by large pharmaceutical companies and by core laboratories that charge the original researcher a sizable amount per slice analyzed. In addition, these techniques are inefficient for identifying proteins in nearby slices because the cutting out of the polypeptide bands from the gel must be done sequentially, and the excision process may shift the gel making subsequent excision steps difficult to control. Also, losses occur when the polypeptides adhere to the walls of the tube.
Quantification is typically carried out on the proteins either before or after they are separated on the gel. There are three methods typically used:    1. Staining with Coomassie or silver stain or fluorescent dyes: At the present time this requires the soaking of the gel in a solution containing protein staining compounds that are visualized in visible light or ultraviolet light after washing away the excess dye from the gel, a process that typically takes tens of minutes to hours.    2. Tagging proteins before separation: Fluorescent proteins derivatives can be made by reacting the proteins with fluorescent dyes prior to separation on a gel. The nature of the fluorescent dye may alter the mobility of the protein derivative during separation on the gel, however, thereby resulting in error. After separation, the fluorescence intensity is measured as described, for example, by Berggren et al., Proteomics 1: 54–65 2001; and Tonge et al., Proteomics 3: 377–396 2001.    3. Tagging proteins with radioactive isotopes: Radio-labeling of proteins prior to separation is commonplace. Visualization is typically done by exposure of the gel containing the separated radio-labeled proteins to photographic film. The amount of exposure is relative to the amount of radioactivity on the protein. However, use of radioactive tags is undesirable because of regulatory considerations regarding access to, exposure to, and disposal of radioactive materials. In addition, many proteomic samples (e.g., serum samples) can not be biosynthetically labeled, and additional chemistry must be performed to introduce radioactivity into the sample.
All three of the foregoing methods require additional steps to stain, destain, wash, and image, which means that more time is required to obtain the results. Quantitative measurements require comparisons to standard proteins labeled in the same fashion and separated in the same manner; thus additional experiments need to be done.
Recently, due to increases in the resolution of matrix assisted laser desorption ionization-time of flight mass spectrometers (MALDI-TOF MS), it has become possible to use heavy stable isotopes for quantitative purposes. In these experiments, a control sample contains proteins that are labeled with normal amino acids, whereas the experimental sample contains proteins that contain heavy isotope enriched atoms (or vice versa), particularly deuterium, C-13, N-15 and O-18 (see Veenstra et al., J Am Soc Mass Spectrom 11: 78–82 2000). The two samples are mixed together prior to protein or peptide separation, and then distinguished by the mass spectrometer. Alternatively, the heavy atoms are added to specific amino acid side-chains by using protein modification reagents that have come in two different isotopic forms as described by Gygi et al, Nat. Biotechnol 17: 994–999 1999. In this case, the chemical modification step can take place either before or after protein digestion. In many cases, isotope enrichment strategies have avoided gels altogether because of the difficulties of the sample processing steps described above. Instead, the proteins are digested as one sample prior to peptide separation, and protein identification is performed at the level of individual peptides, using MS—MS techniques. This technology is not commonly used when the goal is to determine the relative quantification of a limited number of proteins because of reasons of expense. In addition, all information about the starting molecular weight of the proteins is lost when the proteins are digested at the beginning of the experiment, and intact molecular weight is often of crucial interest to biochemists.
A second, unrelated protein analysis technique involves the direct identification of proteins from whole tissues or colonies. In the past, proteins were typically extracted from colonies, tissues and tissue slices by physical disruption of the cells that make up the sample. However, as disclosed in U.S. Pat. No. 5,808,300, it is now possible to analyze proteins that are ablated directly from tissues by mass spectrometry. However, intact proteins are nearly impossible to identify compared to their peptide counterparts.
Recently, a technique for effecting parallel protein identifications, called the molecular scanner, that does not require gel staining, spot excision and extraction and peptide isolation was described in U.S. Pat. No. 6,221,626. An electroblotter sandwich was described comprising a hydrophilic membrane containing an immobilized protease positioned between an electrophoresis gel and a hydrophobic collection membrane which absorbed peptide fragments. The hydrophobic membrane was later treated with a MALDI ionizable matrix that permitted MALDI analyses of the cleaved polypeptide immobilized samples directly from the membrane. Thus all of the proteins that were present in the gel are processed simultaneously using techniques that are compatible with mass spectrometry. In this patent, the electrical current used to drive the peptide fragments to the collection membrane was either pulsed or was alternating current, which required special electroblotting equipment. In addition, this mode of electrical current use is undesirable since it increases the time needed to process samples. This technique was subsequently refined as described in published PCT patent application WO 00\45168 to include an in-gel digestion step. However, neither of these two techniques was completely satisfactory for certain applications. For example, there were technical difficulties in obtaining consistent and sensitive mass signals using the original techniques described above. Also, no techniques were described that allowed simultaneous protein identification and the use of heavy isotopes for protein quantification. These limitations have made it both less desirable and more difficult for biochemists to use the molecular scanner on a casual basis. Both the '626 patent and the published PCT patent application WO 00/45168 described above are incorporated herein by reference.
Accordingly, it would be desirable to provide a method and apparatus for performing molecular scanner experiments that do not require any specialized equipment, so that this technique can be applied to routine biochemical analyses. In addition, it would be desirable to develop a means to use heavy isotopes for protein quantification without losing all information about the protein's starting molecular weight in the sample. The method and apparatus that we describe below fulfills these requirements.